The present invention relates to pharmaceutical preparations useful in the treatment of diseases characterized by a pathological immune response, and processes preparing such pharmaceutical preparations.
Diseases characterized by pathological immune responses include a large number of diseases which are associated with significant mortality and morbidity, and for which no satisfactory treatments are available. Such diseases particularly include auto-immune diseases, such as rheumatoid arthritis (RA) or inflammatory bowel disease (IBD), transplantation-related diseases such as graft-versus-host disease (GVHD).
These diseases result from a conflict of the immune system in which tolerogenic mechanisms controlling homeostasis have been disrupted. Many factors can be a source of such diseases, notably environmental factors, genetic predispositions as well as unresolved inflammation. Indeed, immune cells have the ability to sense bacteria, viruses and other foreign antigens and mount a strong immune response on the one hand, and on the other hand are able to sense self-derived antigens and tolerate them.
In addition to fighting infections, the immune system has the role of maintaining the normal state of health and function of the body. Therefore, throughout the life span of an organism, tissues become reshaped with areas of cells being removed. This is accomplished by a process termed programmed cell-death or apoptosis, the apoptotic cells disintegrate in an orderly and harmless fashion and are phagocytosed. This process of apoptosis is considered to be particularly important in the development and maintenance of the immune system itself, where immune cells which recognize or attack normal cells of the body are destroyed and removed by this process.
Cells which eliminate such unwanted cells are called professional phagocytes. Said professional phagocytes are recruited by several mechanisms and eliminate unwanted cells in order to maintain homeostasis and prevent inflammation.
Cells undergoing death by apoptosis have been shown to demonstrate immunomodulatory properties. Apoptosis is a physiological mechanism that eliminates cells in excess or cells that become undesirable without inducing an inflammatory response. The lack of inflammation associated with apoptosis is attributed to the fact that professional phagocytes, such as macrophages and immature dendritic cells efficiently eliminate apoptotic cells or blebs, thus preventing the release of proteases and other inflammatory mediators such as alarmines, and secondary necrosis of apoptotic cells, another proinflammatory event. This process of efficient apoptotic cell elimination is associated with mechanisms to prevent the initiation of immune responses. These mechanisms are critical and act in parallel since they should prevent the occurrence of autoimmune diseases. Two types of mechanisms can be distinguished: those related to apoptotic cells and those that are related to their elimination by phagocytes.
It has been reported that apoptotic cells, during the process of apoptosis, secrete immunosuppressive cytokines such as IL-10 and TGF-β. TGF-β, stored in a latent form in intracellular compartments, is released during the apoptotic process. These cytokines help to generate an immunosuppressive microenvironment, inhibit the secretion of proinflammatory cytokines (TNF-α or IL-1β) by macrophages and neutralize the induction of an effective immune response. This prevents the initiation of an immune response to self-antigens and prevents autoimmune response development. Apoptotic cells bind inflammatory chemokines such as CCL3 and CCL5 through the expression of CCR5, thus preventing the attraction of other leukocytes. These mechanisms are transient until apoptotic cells are eliminated/engulfed.
Recent publications showed that apoptotic cells also indirectly allow the induction of an immunomodulatory microenvironment through professional phagocytes. Indeed, phagocytes such as macrophages can release, or express immunosuppressive molecules (IL-10, TGF-β, prostaglandin E2, or PGE-2, Fas ligand) during the clearance of apoptotic cells (Griffith, T S and Ferguson, T A, Immunity (2011) 35: 456).
These phagocytes exert immunomodulatory effects to prevent the development of deleterious immune responses to the host. In addition, phagocytes eliminating apoptotic cells also spread this immunomodulatory message to adaptive immunity cells. Macrophages seem to be the main effective phagocytes that remove apoptotic cells.
Indeed, they express a large number of membrane receptors involved in this elimination. Stimulation of these receptors participates in the immunomodulatory properties associated with the removal of apoptotic cells. Numerous immunomodulatory mechanisms have been reported. They are characterized mainly by the release of soluble factors, cytokines such as IL-10 or TGF-β. The response of macrophages to danger stimuli is inhibited once they have removed the apoptotic cells. Certain subpopulations of dendritic cells are also involved in the capture of apoptotic cells. One study conducted on rats, showed that a subpopulation of circulating dendritic cells are responsible for continuously capturing apoptotic cells and bodies from intestinal epithelial cells removed each day after desquamation. Then, the dendritic cells migrate to the mesenteric lymph nodes where they inactivate autoreactive naïve T cells. Other studies, in the mouse model, also suggest that the capture of apoptotic cells by dendritic cells leads to tolerance. Moreover, an in vitro study showed that dendritic cells that have captured apoptotic cells did not respond to lipopolysaccharide (Morelli, A E and Larregina, AT, Apoptosis (2010) 15: 1083).
The consequences of apoptotic cell/phagocyte interaction influence the polarization of CD4+ T cells. Although contact with apoptotic cells inhibits the maturation and cytokine production of conventional dendritic cells, their migration capabilities are not affected. Thus, these cells can acquire CCR7 expression and migrate in response to gradients of CCL19 and CCL21 to the lymph nodes closest to the site where the cells die. In lymph nodes, dendritic cells having seen apoptotic cells can interact with naive CD4+ T cells and therefore favor their conversion to a regulatory phenotype such as regulatory T cells (Treg) through an immunomodulatory “message”. A major characteristic of Treg generated by “immunomodulatory” dendritic cells is their ability to increased IL-10. It was also suggested that plasmacytoid dendritic cells are antigen presenting cells at the origin of Tr1 cells. However, these dendritic cells can also promote the differentiation of FoxP3+ Treg, notably in an apoptotic cell-rich microenvironment.
Traditional methods to treat diseases characterized by a pathological immune response with immunosuppressive drugs such as corticosteroid, azathiopine, cyclophosphamide, methotrexate and cyclosporine are far from being ideal treatments. The drug therapy is accompanied by serious side effects, including general immune suppression, leading to high rates of morbidity and being the primary cause of premature mortality. Furthermore, currently used immunosuppressive drugs have shown little or no impact on chronic rejection such as allograft rejection and graft-versus-host disease and therefore on overall long-term allograft survival. Importantly, long-term pharmacological immunosuppression is associated with toxicity and increased incidence of malignancies and infectious and metabolic diseases.
Therefore, the discovery of alternative ways for the manipulation of the immune system to treat such immune diseases has been a major goal for many years.
Apoptotic cells favour an immunosuppressive microenvironment through several mechanisms as described above. Therefore, several strategies using apoptotic cells for manipulating the immune system have already been proposed. The patent application WO2006/117786, for example, discloses injections of a cell preparation comprising dying or dead leukocytes to treat diseases associated with pathological immune responses.
However, the injection of cells suffers from various drawbacks such as the requirement for administration of allogeneic apoptotic cells, which is associated with the risk of allo-immunization favoring graft rejection since apoptotic cells have been demonstrated as an source of allo-antigens, and the failure to demonstrate adequate safety with respect to potential for inflammatory side-effects.
The patent application WO2010/070105 discloses an approach supposed to circumvent the problem of injecting cells into a subject. The application suggests using the supernatant of apoptotic cells to manipulate a subject's immune system. Nevertheless, recent research showed that the supernatant of apoptotic cells alone fails to activate for example plasmacytoid dendritic cells which are important to favor tolerance induction by mechanisms including regulatory T-Cell (Treg) induction (Bonnefoy F et al. J Immunol. 2011;186(10):5696-705).
The patent WO2007/0559922 addresses this aspect and uses supernatant of apoptotic cells ex-vivo in order to generate regulatory T-cells which are then administered to the subject in need, especially subjects suffering from graft-versus-host diseases. Nevertheless, this application also suffers from drawbacks associated with the injection of foreign cell material into a subject, such as potential for inflammatory side-effects.
Thus, all the previous approaches have failed to provide standardized and efficient treatment of such diseases. Indeed, the therapeutic effect of apoptotic cell injection is strongly associated with apoptotic cell removal, therefore depending on the migration of apoptotic cells to phagocytic tissues, efficiency of phagocytes to remove apoptotic cells and of importance, the ability of phagocytes to generate such a tolerogenic environment.
Thus, it would be highly advantageous to propose a pharmaceutical preparation independent of such limitations.